The slime coatings of serpentine via its hydrophobic Si-O layers significantly weaken the floatability of the hydrophobized sulfide minerals. In this paper, sodium lignosulphonate (SLS) was adopted to avoid the aggregation of fine serpentine particles on to the hydrophobized pyrite surface. The micro-flotation findings indicated that for the artificial mixture of pyrite and serpentine (2:1 mass ratio), 1.0 × 10–4 mol/L sodium isobutyl xanthate (SIBX) only floated out ∼22 % pyrite at pH ∼9.0, while, after extra addition of 70 mg/L SLS, the floatability of pyrite was restored and its recovery reached ∼90 %. In situ AFM (Atomic Force Microscope), contact angle, zeta potential, fourier transform infrared spectrometer (FTIR) and optical microscope deduced that the strong affinity of SIBX to pyrite rendered it replace the most part of the pre-aggregated SLS from pyrite surface, thus to restore the hydrophobicity of pyrite and further decrease the zeta potential. In addition, SLS attached on the positively-charged Mg sites of serpentine through its carboxyl and phenolic hydroxyl groups reduce the hydrophobicity and zeta potential of serpentine. While, the hard base O atom/anion exhibited much stronger affinity to hard acid Mg cation than that of the soft base S atom/anion, SIBX hardly replaced the adsorbed SLS from serpentine surface. Therefore, the increased electrostatic repulsion and weakened hydrophobic attraction between the SLS-adsorbed serpentine and SIBX-hydrophobized pyrite avoided their aggregation. The combination of SLS and SIBX realized the selective flotation separation of pyrite from serpentine.